LOCUS       sequence-249725-        6948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  COL4A3 mutation-specific sgRNA under the control of U6 promoter; 
            mCherry-sgRNA target-(out of frame)EGFP expression cassette; COL4A3 
            donor template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6948)
  AUTHORS   Daga S, Donati F, Capitani K, Croci S, Tita R, Giliberti A, 
            Valentino F, Benetti E, Fallerini C, Niccheri F, Baldassarri M, 
            Mencarelli MA, Frullanti E, Furini S, Conticello SG, Renieri A, 
            Pinto AM
  TITLE     New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene 
            editing in podocyte-lineage cells.
  JOURNAL   Eur J Hum Genet. 2019 Nov 21. pii: 10.1038/s41431-019-0537-8. doi: 
            10.1038/s41431-019-0537-8.
  PUBMED    31754267
REFERENCE   2  (bases 1 to 6948)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Eur J Hum 
            Genet. 2019 Nov 21. pii: 10.1038/s41431-019-0537-8. doi: 
            10.1038/s41431-019-0537-8."
COMMENT     Sequence Label: pAAV_Donor/Reporter-COL4A3
FEATURES             Location/Qualifiers
     source          1..6948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   248..377
                     /label=AAV2 ITR
     enhancer        643..946
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        947..1150
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1101..1121
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1187..1894
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1332..1350)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1990..1999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1996..1998,1999..2001,2002..2715)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1996..1998,1999..2001,2002..2712)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2041..2062)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2302..2321)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2649..2670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2788..2808)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3206..3217)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3284..3301)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3290..3497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3628..3709
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3665..3684)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3726..3974
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3726..3746
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3897..3916
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3996..4071
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   4255..4384
                     /label=AAV2 ITR
     primer_bind     complement(4403..4420)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4403..4419)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4412..4434)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4561..5016
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4648..4667)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4858..4879
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5043..5147
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5148..5216,5217..6008)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5366..5385)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6179..6767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6668..6687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc
      301 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      361 catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc
      421 atgctctagg aagatcggaa ttcgccctta aagacccagg aattccaggc ttggatagat
      481 caggatttcc tggagaaact ggatcaccag gaattccagg tcatcaaggt gaaatgggac
      541 cactgggtca atcgacggta tcgaattcgc ccttaagcta gctagttatt aatagtaatc
      601 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      661 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      721 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      781 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      841 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      901 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      961 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     1021 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     1081 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     1141 aagcagagct ggtttagtga accgtcagat ccctagcgga ttcaccatgg tgagcaaggg
     1201 cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg
     1261 ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg
     1321 cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat
     1381 cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc
     1441 cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga
     1501 ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta
     1561 caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaaaaagac
     1621 catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga
     1681 gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac
     1741 ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga
     1801 catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg
     1861 ccactccacc ggcggcatgg acgagctgta caagaagctt cgtctctgtc gcctggagaa
     1921 actgaatcac ctggtctgcg agacggaatt ctgcaatcga cggtaccacg ggcccgggat
     1981 ccaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2041 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2101 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2161 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     2221 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2281 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2341 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2401 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2461 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2521 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2581 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2641 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2701 gagctgtaca agtaaagcgg ccgcgcttgg atccaatcaa cctctggatt acaaaatttg
     2761 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     2821 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     2881 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     2941 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     3001 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     3061 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     3121 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     3181 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3241 cctgctgccg gctctgcggc ctcttccgcg tcttcgagat ctgcctcgac tgtgccttct
     3301 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3361 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3421 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3481 agcaggcatg ctggggactc gagttagcgg ccgcgactct agatcataat cagccatacc
     3541 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     3601 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     3661 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgc
     3721 cgcgggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     3781 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     3841 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     3901 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     3961 aaggacgaaa caccgcctgg agaaactgaa tcaccgtttt agagctagaa atagcaagtt
     4021 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
     4081 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
     4141 ctgcagacaa atggctctag aggtaccctc gagttaaggg cgaattcccg attaggatct
     4201 tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
     4261 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     4321 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     4381 gcagccttaa ttaacctaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4441 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4501 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     4561 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4621 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4681 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4741 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4801 catcgccccg atagacggtt tttcgccctt tgacgctgga gttcacgttc ctcaatagtg
     4861 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4921 aagggatttt tccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4981 acgcgaattt taacaaaata ttaacgttta taatttcagg tggcatcttt cggggaaatg
     5041 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5101 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5161 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5221 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5281 tcgaactgga tctcaatagt ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5341 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5401 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5461 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5521 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5581 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5641 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagtaatgg
     5701 taacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5761 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5821 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5881 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5941 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6001 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6061 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6121 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6301 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     6361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6781 cggccttttt acggttcctg gccttttgct gcggttttgc tcacatgttc tttcctgcgt
     6841 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6901 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
